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Graduate training in cellular

Thomas D. Pollard Department of and , Johns Hopkins , Baltimore, Maryland 21205 USA INTRODUCTION In this article I will outline some of the knowledge and Good problems focus on important biological pro- skills that investigators in cellular biophysics find valu- cesses, offer growth potential, and are tractable experi- able in their work. Everyone has their own definition of mentally. Some newcomers imagine that previous gener- cellular biophysics. For me it is hard to separate cellular ations have already grabbed all ofthe good problems, but biophysics from , so let us accept it is not true. The landscape is so vast in biological re- the inevitable overlap and differentiate the areas on the search that new knowledge creates new opportunities. As basis of emphasis. Both include quantitative studies of the frontiers broaden, so will the opportunities, at least the chemical and physical basis of cellular . for the foreseeable future. Not only are there essentially Both involve characterization of , nucleic acids, limitless details awaiting discovery; even more impor- and and their interactions. But a molecular bio- tant, many ofthe general principles still need to be iden- is more likely to be interested in electrons, tified. while a cellular biophysicist is more interested in macro- "Important biological processes" are those that cells molecular events that can be studied with a micro- depend on for their basic function. Some of the best scope. Cellular biophysics emphasizes the molecular problems deal with universal processes used by all cells aspects that directly illuminate functions at the cellular such as the assembly oforganelles, motility, signal trans- level, while molecular biophysics emphasizes the proper- duction, and regulation of the cell cycle, but the parsi- ties ofthe themselves. They really represent a mony ofmechanisms at the molecular level has the con- continuum with a common goal. sequence that investigation of specialized processes I write from the perspective of almost 25 years of re- usually reveals some basic principles with wide applica- search on the molecular basis ofcellular motility, an en- bility. For example, the wealth ofinformation on muscle deavor that started out as a purely biological fascination contraction has greatly enriched our understanding ofall and has taken on a strongly biophysical tone as our un- sorts of cellular motility. derstanding has broadened. I have a very practical view There are so many genuinely fundamental questions of training in cellular biophysics, since as an MD I had in biology that no one should waste their time repeating essentially no formal training in the subject. Instead, my established work in new or with new methods students, postdocs, and I have learned on the job from unless they have an attractive hypothesis about why such the literature, books, and many generous colleagues. I repetitive work is likely to reveal new principles of gen- have also had the opportunity to learn what challenges eral interest. The scientific responds more students while teaching a laboratory course during the enthusiastically to something novel. Discovery not only summer at Woods Hole. Consequently, I will propose provides the most gratification in research, but discovery what one needs to know to do creative research rather is the only way to gain the confidence ofthe community than what constitutes a formal course ofstudy. Although that is required to obtain the resources to continue our knowledge ofthis material will not replace a good idea, it research. One must not waste good training and re- does free one's thoughts from the constraints of igno- sources on boring problems. rance and can lower one's natural inhibitions to try new There is another side to this argument espoused by my things. Good ideas can come from many places, so I put friend Evan Eisenberg of the NIH. When we were post- a very high priority on developing broad interests in biol- docs together, he claimed that the best way to become ogy and a lifetime of reading widely about the natural famous is to identify the most important experiment in sciences. Ostrich-like individuals with their heads stuck one's field and to repeat it. Of course, he did not mean in the sand oftheir own subspecialty risk becoming fossil- that one should literally repeat earlier work; rather one ized. should try a creative variation of the important experi- ment in the hopes of expanding knowledge at the heart ofa field. This approach does not have the novelty that I WHAT IS A GOOD BIOLOGICAL PROBLEM? have advocated above, but it does focus one's attention Formal curricula almost always neglect the first and on the important issues in a field. Since experiments are most important prerequisite for successful research, a rarely replicated exactly, Evan is right that one has a good biological problem. Lest we not forget it here, I will good chance of finding something new that will com- start by considering some ofthe features ofa good biolog- mand attention simply because of the focus on an issue ical problem from my perspective as a cell . of acknowledged importance.

14501 450 0006-3495/92/11/1450/05 $2.00 Biophys. J. © Biophysical Society Volume 63 November 1992 1450-1454 By "growth potential" I mean that one's hard work are not essential for most work in cellular biophysics. should be rewarded by expanding opportunities, not Some expertise with computers is essential, but I hesitate dead ends. Most research requires a substantial invest- to recommend a formal course, since most individuals ment of thought, time, effort and financial resources. learn their computing skills informally on the job. Ideally, each of our investments in research should open up new opportunities. Common examples include the Biology discovery of a new process or , the determina- The aspiring cellular biophysicist needs to have a broad tion of a new structure, the identification and cloning of knowledge of biology to help identify a good research a new gene, the purification ofa new cellular constituent, problem and the most advantageous experimental sys- or the development of a new method. These contribu- tem to get the answers. Courses in general biology are tions can elevate a field to a higher level that allows work necessary for exposure to the diversity oforganisms and to expand in more sophisticated ways. In many cases for orientation, but are not adequate for appreciating the students or postdoctoral fellows have based their inde- merits of a research project. One needs to understand in pendent careers on the hard work they did to elevate some depth how cells work at the molecular level. The their field. Obviously, one should not leave loose ends best way is probably a semester or year long course in cell behind, but the scientific community places such a high biology. Either in this course or others, one must learn value on novelty, that we all should strive to find some- about classical and . Many thing new. biophysicists deal with topics like membrane channels By "tractable" I mean experimentally approachable. that are covered in courses, another impor- Many important problems with growth potential are not tant option to consider. Each student will need to be easy or someone would have taken them on already. selective in choosing biology courses since the topics cov- What is usually missing is a good idea about what to do ered in , for example, differ widely from or how to get started, rather than the absence ofthe tech- school to school. nology to do the work. Many fields are idea limited What about an aspiring cellular biophysicist who rather than technology limited. How many times have comes from the physical sciences with little or no oppor- you said, "I wish that I had thought ofthat"? Since con- tunity for formal training in biology? I would strongly siderable physical labor is usually required to do novel recommend that they read and think about the material research with growth potential, be sure that you have in one ofthe big, comprehensive cell biology books such done some good thinking ahead oftime. Make sure that as the "Molecular Biology of the Cell" by Alberts et al., your strategy is sound and that the outcome will meet 2nd ed. (1989, Garland Press, New York) or "Molecular your goals of contributing something novel and giving Cell Biology" by Darnell, Lodish, and Baltimore, 2nd you the desired foothold for your future work. ed. (1990, Freeman Publications, San Francisco). Both Who should pick the experimental problem and the of these books cover cellular biology at the molecular research strategy? It must be the person with the best level in enough depth to provide an excellent basis for idea. Since no one has a monopoly on good ideas, these thinking about cellular biophysics. One shortcoming is decisions turn out to be team efforts. Ideally everyone in that neither book consistently highlights the limits ofour a lab is continuously hunting for good ideas. The group knowledge, so a naive reader is likely to get the impres- leader may have the best perspective, but everyone sion that there are more answers than we actually have in should contribute. In our laboratory the technical staff, hand. students, and postdocs have all contributed to the overall direction of the research in addition to providing solu- tions to the everyday experimental problems. The only Every cellular biophysicist needs a good working knowl- way for this synergism to develop is to have everyone edge ofbiochemistry, since virtually all experimental bi- informed about the details of their colleague's experi- ologists are , at least in part. An upper divi- ments through give and take at frequent laboratory meet- sion or graduate level biochemistry course is adequate to ings. equip the cellular biophysicist with a working vocabu- lary and the general concepts. This will suffice for formal training, providing that students retain an active interest FORMAL COURSES in the field and do their best to keep up with trends and I will assume that all students entering cellular biophys- new discoveries. Few will master biochemistry without a ics will have a general background in science and mathe- formal course, although thorough study of one of the matics. For undergraduates, this will include courses in major biochemistry books is a possible alternative. The general , , and , each books by Stryer (3d., 1988, W. H. Freeman and Co., with a laboratory. This trio along with calculus is gener- New York) and Matthews and vanHolde (1st ed., 1991, ally required for the advanced courses considered below. Benjamin/Cummings Publishing Co., Redwood City, Upper level courses in physics and math may be helpful CA) are examples of the excellent biochemistry books for some types of research in molecular biophysics but that could be studied independently.

Pollard Training in Cellular Biophysics 1451 One does not have to learn to operate each type of Every cellular biophysicist also needs solid grounding in microscope, but all cellular biophysicists should know , but it is not obvious where they the capabilities ofthe range ofmodern microscopes, be- should get this formal training. The problem is that cause this knowledge will help with the selection of the many physical chemistry courses for emphasize appropriate microscope for each application. It is useful topics and approaches that are not particularly helpful in to understand the principles of bright field, dark field, cellular biophysics. Coverage ofphysical chemistry with polarization, phase contrast, , and differen- a sciences thrust is recommended providing that this tial interference microscopes, ofbasic image formation, does not mean watered down physical chemistry. Alter- video cameras, and video image processing, of confocal natively, some biophysical chemistry classes have the laser scanning microscopes, and of transmission and right orientation, but they do not necessarily cover the scanning electron microscopes. Scanning probe micros- essential topics. The most important topics are thermo- copy is already a valuable tool in and dynamics, , (including will have an impact in biology once specimen prepara- scattering), , andtransport processes (hydrodynam- tion methods are mastered (see A. Engel, 1991, Annu. ics). Quantum and statistical are less useful in Rev. Biophys. Biophys. Chem. 20:79-108). Each type of cellular biophysics than the other topics, but will be valu- involves specialized methods of specimen able for selected students whose research involves these preparation, especially electron microscopy, and it is tools. The optics should include diffraction, lenses, helpful to know in general what they involve. image formation, and image processing as they apply to Many research institutions have microscopy facilities visible light, x-rays, and electrons. The student needs to that provide practical training in microscopy and speci- understand general principles like the relation of lenses men preparation. Good facilities will also offer theory to to Fourier transforms and microscopy to diffraction go with the practice. For advanced training intense methods rather than the intricacies ofany ofthese meth- courses in microscopic theory and practice are available ods. For those who need to study the material on their at the Marine Biological Laboratory, Woods Hole, MA. own or who need a refresher, I like "Physical Chemistry Purification and manipulation of DNA, with Applications in the Life Sciences" by D. Eisenberg proteins, lipids and D. Crothers (1979, Benjamin-Cummings Publish- ing Co., Redwood City, CA). Another good book is To do molecular studies on cells, one must know the Barrow's "Physical Chemistry for the Life Sciences" theory and practice ofhandling the major biological mol- (1975, McGraw-Hill Inc., New York). ecules: at least proteins and nucleic acids, but some knowledge of lipids can also be useful. One needs to know how to purify, manipulate, and analyze these mole- THEORY AND PRACTICAL KNOWLEDGE cules. In the case ofproteins, this requires centrifugation, , electrophoresis, amino acid analysis, Microscopy and sequencing. For DNA this requires library Microscopy of all sorts is probably the most universally construction, cloning, sequencing, in vitro mutagenesis, applicable technique in cellular biophysics, so it is regret- DNA and RNA blots, and expression in bacteria and table that many users do not fully understand how micro- eukaryotic cells. For lipids this requires extraction, col- scopes work. I suppose that the problem is that none of umn chromatography, thin layer chromatography, gas the standard courses regularly takes responsibility for mi- chromatography, and the formation of vesicles and mi- croscopy. Basic optics and the properties oflight are cov- celles. Most of this material is covered in graduate bio- ered in elementary physics, but image formation, meth- chemistry courses, but the level is usually much too su- ods ofenhancing contrast, and quantitative fluorescence perficial to be of practical use. Individuals in most insti- methods are not. The student usually has to shop around tutions and many laboratories are familiar with these to find some instruction in microscopy. Fortunately, methods, but I advise newcomers to be cautious about much of the basic theory is covered in books including their sources of advice, since many successful practi- the following: "Fundamentals ofLight Microscopy" (M. tioners do not fully understand the methods that Spencer, 1982, Cambridge University Press, New York); they use. "Video Microscopy" (S. Inoue, 1986, Plenum Press); Fortunately, many excellent books are available on "Fluorescence Microscopy of Living Cells in Culture" biochemical methods, for example, "Protein Purifica- (Y. L. Wang and D. L. Taylor, editors, 1989, volumes 29 tion" by R. K. Scopes (1987, Springer-Verlag, New and 30 ofMethods in Cell Biology, Academic Press, New York) for proteins, "Molecular Cloning" by Sambrook York); "Biological Confocal Microscopy" (J. Pauley, edi- et al. (2nd ed., 1990, Cold Spring Harbor Laboratory, tor, 1989, Plenum Press, New York). A good source of Cold Spring Harbor, NY) for manipula- information on fluorescent probe molecules is the cata- tions, and "Techniques ofLipidology", M. Kates (1986, logue "Molecular Probes" (R. Haugland, 1992, Molecu- Elsevier Scientific Publishers, Amsterdam) for lipids. lar Probes Company, Eugene, OR). The series "Methods in Enzymology" (Academic Press,

14521452 Biophysical JournalJoumal Volume 63 November 1992 New York) has covered all of these methods in detail the relation of rate constants to equilibrium constants, over the years. I strongly recommend that you consult and the experimental strategies used to evaluate rate these or more advanced books the first time that you use constants. These include stopped flow and quenched a new method to give yourselfthe benefit offully under- flow rapid kinetics as well as single turnover and pulse- standing the principles and the practical matters in- chase designs that are applicable to virtually every biolog- volved. If you do this reading consistently, you will soon ical system. A laboratory section on kinetics is available become the expert in your lab on methods, since most as part ofthe Physiology Course at the Marine Biological people only have hit or miss, word of mouth training in Laboratory. many ofthe techniques upon which their work depends. Although it is no longer usually included in the list of Spectroscopy skills in handling , I think that it is es- sential for everyone working with pure macromolecules The cellular biophysicist must be knowledgeable about to know the methods available for crystallization. The the theory and practice ofUV-visible spectroscopy, fluo- individual who does the purification is most likely to rescence spectroscopy, and simple light scattering. Some know the tricks required to keep a particular molecule of this is purely mechanical: how to take an absorption happy during a crystallization trial, so they, rather than a spectrum, how to choose the optimal wavelengths for a professional crystallographer, should take the first crack fluorescence measurement, what sample concentrations at growing crystals suitable for structure determination give reliable measurements, how to increase the signal- by x-ray analysis. I claim that the actual manipulations to-noise ratio. For fluorescence work, this usually re- are much easier than running a gel and the hardware quires an understanding of the chemical methods and required to set up some hanging drops for vapor diffu- basic fluorescent reagents available to label specific sites sion crystallization trials is less expensive than lunch at a on biological molecules with fluorescent probes. Pho- fast food restaurant. A detailed book by A. MacPherson toactivated fluorescence probes have recently emerged on "Protein Crystallization" (1982, John Wiley and as particularly valuable probes. Sons, Inc., New York) is available for practical advice. It is also extremely useful to understand enough about An intense practical course in x-ray is the theory and practice of specialized spectroscopic available annually at the Cold Spring Harbor Labora- methods so that one knows when and how to consult an tory. expert colleague for a particular experimental applica- tion. The most important advanced methods are x-ray Handling cells diffraction and multidimensional NMR methods for atomic structure determination. At some time in our ca- Good sterile technique is required for both culture reers, each ofus is likely to find a valuable application for and bacteriology and thus for everyone who clones other specialized types of spectroscopy including ESR, DNA, expresses cloned proteins and prepares live cells NMR, IR, CD, mass spectroscopy, quasielastic light for microscopy. More specialized techniques, including scattering, phosphorescence, fluorescence trans- cellular microinjection, intracellular recordings, and fer, fluorescence life time, and fluorescence polarization, patch clamping, are the mainstays ofsome types ofcellu- so it is useful to know what they have to offer. lar biophysics. A general knowledge of these valuable methods will suffice for the undifferentiated cellular bio- physicist. Practical courses are available at most universi- KEEPING UP ties, the Marine Biological Laboratory and the Cold Spring Harbor Laboratory. Keeping up with advances in research is probably the most challenging task for most practicing biological sci- entists. Having become acquainted with biology in gen- Kinetics eral, having mastered a particular field and (hopefully) Every cellular biophysicist should understand how to use having contributed something original, how should the reaction rates to determine mechanisms. The standard cellular biophysicist keep up with the literature? Obvi- steady-state kinetics covered in biochemistry ously, one must read carefully the emerging literature in classes is one way to look at the subject, but is not nearly one's own specialty, that goes without saying. Each ofus as useful as a general approach as transient state kinetics. knows well the three or fourjournals where we and col- For example, transient kinetics can be used to evaluate leagues in a field are most likely to publish new results. not only enzyme mechanisms in more detail than possi- These new papers need to be read, not just xeroxed. ble with steady-state kinetics, but the same general ap- (Faced with spiralling costs at the copy center at the proach can also be applied to the host of binding reac- Cambridge MRC Laboratory ofMolecular Biology, Sid- tions and conformational changes that cells use to assem- ney Brenner once sent out an appeal to replace xeroxing ble their macromolecular components, to regulate genes with "memeroxing"!) and membrane permeability, and to transduce signals. Since few individuals are disciplined enough to read You need to understand the meaning of rate constants, the original literature on their own, I strongly recom-

Pollard Training in Cellular Bigphysics 1 453 mend specialized journal clubs. A common format is to Broad departmental journal clubs and seminar series are assign important papers on a rotating basis to the also valuable. Since one can read only a tiny fraction of members of the group and have them lead an in depth the original literature, I recommend regular reading of discussion of one or two papers per session. I have had one or more of the excellent review journals such as better results with a literature scanning format. Each of Current Opinion in Cell Biology or Current Opinion in the 10 members of our journal club selects a different (each of which systematically covers paper each week and has 10 minutes on the clock to their whole field each year) or Trends in Biochemical present all ofthe figures in the paper to the whole group. Sciences or Trends in CellBiology(which highlight excit- The papers available each week are those that seem most ing new work). Similar publications are available in ge- interesting to me from a computerized literature search netics and . A second essential reading as- that casts a rather wide net in our field. This format al- signment is to peruse and Science each week. Not lows each participant to see the data from more than 300 much of major importance escapes publication there ei- original papers each year. Although we cannot consider ther as original research or as news features. Finally, each in depth, the participants rapidly learn the skills to teaching (that oft maligned obligation of professors) is provide criticism and praise for their papers each week. probably the best way to stay informed in the general In some ways, this journal club is even more valuable field of cellular biophysics. Courses with biological con- and certainly more cost effective than attendance at sci- tent will provide much more educational benefit to the entific meetings. professor than the all too frequent biophysics courses Keeping abreast with the action outside one's own that methods. field is more challenging, but no less important. It is ad- vantageous to know in general where the action is in the I thank the members of my laboratory for their thoughts on this topic whole field of cellular biophysics, because many of the and Ed Lattman, Shin Lin, and Ted Salmon for their helpful com- ments on the manuscript. Obviously, they not be held responsi- most valuable insights come at the interfaces ofdifferent should ble for the opinions expressed herein. fields of study. Further, one needs to know how investi- gators are thinking in related fields to keep one's own The author's research is supported by grants from the National Insti- ideas fresh. Participation in large national scientific meet- tutes of Health. ings, especially careful attention at the symposium talks outside one's speciality, is an excellent way to keep up. Receivedfor publication and infinalform 13 July 1992.

Volume 63 November 1992 14541 454 Biophysical JoumalJournal Volume 63 November 1992